世界的に重要な腸管病原性細菌を食品から容易に定量検出できる高感度法の開発

2014 Fiscal Year Annual Research Report

• Project/Area Number: 24249038
• Research Institution: Kyoto University
• Principal Investigator: 西渕 光昭 京都大学, 東南アジア研究所, 教授 (50189304)
• Co-Investigator(Kenkyū-buntansha): 中口 義次 石川県立大学, 生物資源環境学部, 准教授 (70378967)
• Project Period (FY): 2012-10-31 – 2015-03-31
• Keywords: 腸炎ビブリオ / 腸管出血性大腸菌 / 免疫磁気ビーズ法 / LAMP / IMS

Outline of Annual Research Achievements

免疫磁気ビーズ（IMS）法およびloop-mediated amplification (LAMP) 法を採用して、腸炎ビブリオの病原性菌株を魚介類中から高感度・簡便・迅速に定量検出する検査法に関して、病原性菌株のうち、tdh遺伝子陽性株については、すでに発表した方法をさらに改良し、温帯および熱帯地域のいずれでも正確な感度で、かつ以前よりさらに簡便に検出できる方法を確立した。trh遺伝子陽性株については、同様の方法を目指して開発を進めてきたが、環境由来株の中に塩基配列の置換のために溶血活性が陰性である菌株がかなり存在することを確認した。そのため、今後溶血活性を示す菌株に特有な塩基配列のみを標的とするLAMP用プライマーを設計し、これを組みこんだ系にして開発を完了する必要があると判断した。
腸管出血性大腸菌 (EHEC) に関しても、同様にIMS法およびLAMP 法を採用した肉類の定性検査法を確立した。ただし、IMS法における標的O抗原はO157のみならず世界的に特に重要であると考えられる15種のうちの12種であるので、重要なnon-O157型EHECをほとんど検出できる。今後はこれをMPN法と組み合わせて定性検査法に発展させると、より実用的であると考えられる。
上記の2菌種に関する検査法は、今年度から検査試薬を乾燥状態のものを採用（高温地域でも安定、結果の肉眼による判断が容易）したので、簡便性が向上した。また、食品中の標的菌濃度が低い日本（温帯の先進国）および高いタイ（熱帯の発展途上国）で実験を実施し、実用性が確認できている。さらに2014年10月には、インドネシアの西スマトラ州パダン市（赤道直下）でワークショップを開催し、腸炎ビブリオに関する検査法を紹介し、賞賛の声をきくことができた。
また、本法の将来応用可能な関連下痢原因菌のアジア諸国における情報も収集できた。

Research Progress Status

26年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

26年度が最終年度であるため、記入しない。

Research Products

• [Journal Article] Development of a method for detection of Shigatoxin-producing Escherichia coli belonging to clinically important twelve O serotypes based on the combination of PickPen-assisted immunomagnetic separation and loop-mediated isothermal amplification (2015)
  • Author(s): Kayali, A. Y. O. Escalante-Maldonado, V. Vuddhakul, K. Seto, Y. Nakaguchi, and M. Nishibuchi
  • Journal Title: Int. J. Immunol. Immunother. Volume: 2(1) Pages: 1-7
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] LAMP assay to detect Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease in shrimp (2015)
  • Author(s): Kongrueng, J., N. Tansila, P. Mitraparp, P. Mitraparp-arthorn, M. Nishibuchi, G. J. Vora, V. Vuddhakul
  • Journal Title: Aquacult. Int. Volume: 無し Pages: 無し
  • DOI: 10.1007/s10499-9874-3
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Detection and quantification of pathogenic Vibrio parahaemolyticus in shellfish by using multiplex PCR and loop-mediated isothermal amplification assay (2015)
  • Author(s): Malcolm , T. T. H., Y. K. Cheah, C. W. J. W. M. Radzi, F. A. Kasim, H. K. Kantilal, T. Y. H. John, J. Martinez-Urtaza, Y. Nakaguchi, M. Nishibuchi, R. Son
  • Journal Title: Food Control. Volume: 47 Pages: 664-671
  • DOI: 10.1016/j.foodcont.2014.08.010
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Most-probable-number loop-mediated isothermal amplification–based procedure enhanced with K antigen–specific immunomagnetic separation for quantifying tdh+ Vibrio parahaemolyticus in molluscan shellfish (2014)
  • Author(s): Tanaka, N., Y. Iwade, W. Yamazaki, F. Gondaira, V. Vdddhakul, Y. Nakaguchi, and M. Nishibuchi
  • Journal Title: J. Food Protect. Volume: 77(7) Pages: 1078-1085
  • Peer Reviewed
• [Journal Article] Vibrio parahaemolyticus and its specific bacteriophages as an indicator in cockles (Anadara granosa) for the risk of V. parahaemolyticus infection in southern Thailand (2014)
  • Author(s): Yingkajorn M., N. Sermwitayawong, P. Palitapongarnpimp, M. Nishibuchi, W. P. Robins, J. J. Mekalanos, and V. Vuddhakul
  • Journal Title: Microbil. Ecol. Volume: 67(4) Pages: 849-856
  • DOI: 10.1007/s00248-014-0382-9
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Effect of detergents as antibacterial agents on biofilm of antibiotics-resistant Vibrio parahaemolyticus isolate (2014)
  • Author(s): Elexson, N., L. Afsah-Hejri, Y. Rukayadi, P. Soopna, H.Y. Lee, T.C. Tuan Zainazor, M. Nor Ainy, Y. Nakaguchi, Nishibuchi Mitsuaki, R. Son
  • Journal Title: Food control Volume: 35 Pages: 378-385
  • DOI: 10.1016/j.foodcont.2013.07.020
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Transfer of Listeria monocytogenes between abiotic surfaces under different weights (2014)
  • Author(s): Lee, H. Y., L. C. Chai, N. A. Mahyudin, M. Nishibuchi, and R. Son
  • Journal Title: Food Sci. Biotechnol. Volume: 23(4) Pages: 1237-1241
  • DOI: 10.1007/s10068-014-0169-2
  • Peer Reviewed / Open Access
• [Journal Article] Quantification of enterophathogenic Escherichia coli from retailed meats (2014)
  • Author(s): Pannuch, M., Sirikaew, S., Nakaguchi, Y., Nishibuchi, M., Sukhumungoon, P.
  • Journal Title: Food Research Journal Volume: 21(2) Pages: 547-551
  • Peer Reviewed / Open Access
• [Journal Article] First report in Thailand of a stx-negative Escherichia coli O157 strain from a patient with diarrhea (2014)
  • Author(s): Themphachana, M., Y. Nakaguchi, M. Nishibuchi, K. Seto, P. Rattanachuay, K. Singkhamanan P. Sukhumungoon
  • Journal Title: Southeast Asian J Trop Med Public Health Volume: 45(4) Pages: 1-9
  • Peer Reviewed
• [Journal Article] Consumption of raw oysters: A risk factor for Vibrio parahaemolytcus infection (2014)
  • Author(s): New, C. Y., H. K. Kantial, M. T. H. Tan, Y. Nakaguchi, M. Nishibuchi, and R. Son
  • Journal Title: Int. Food Res. J. Volume: 21(6) Pages: 2459-2472
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Presentation] 世界から腸炎ビブリオ感染症をなくすという夢は現実となるか？ (2015)
  • Author(s): Mitsuaki Nishibuchi
  • Organizer: 第88回日本細菌学会総会ワークショップ「変遷する食中毒」
  • Place of Presentation: 長良川国際会議場、岐阜
  • Year and Date: 2015-03-26 – 2015-03-28
  • Invited
• [Presentation] Improvement of IMS-LAMP-based Detection Method for Pathogenic Vibrio parahaemolyticus from Seafood (2015)
  • Author(s): Oscar R. Escalante-Maldonado1, Ahmad Yaman Kayali, Wataru Yamazaki, Fumio Gondaira, Yoshitsugu Nakaguchi, and Mitsuaki Nishibuchi
  • Organizer: 第88回日本細菌学会総会
  • Place of Presentation: 長良川国際会議場、岐阜
  • Year and Date: 2015-03-26 – 2015-03-28
• [Presentation] Optimization of the LAMP-IMS-based detection method for detecting enterohemorrhagic Escherichia coli from beef (2015)
  • Author(s): Ahmad Yaman Kayali, Oscar R. Escalante-Maldonado, Fumio Gondaira,
  • Organizer: 第88回日本細菌学会総会
  • Place of Presentation: 長良川国際会議場、岐阜
  • Year and Date: 2015-03-26 – 2015-03-28
• [Presentation] How can the area study collaborate with other study fields? －A successful collaboration between the area study respecting “multidisciplinary ” and ”glocal” concepts and a micro-scale analyses-orientated modern biomedical study (2015)
  • Author(s): Mitsuaki Nishibuchi
  • Organizer: Kyoto U - UC San Diego Joint Symposium
  • Place of Presentation: ANN Crown Plaza, Kyoto, Japan
  • Year and Date: 2015-03-11 – 2015-03-12
  • Invited
• [Presentation] Improvement of the MPN-IMS-LAMP- based Detection Method for Virulent Strains of Vibrio parahaemolyticus from Seafood (2015)
  • Author(s): Oscar R. Escalante-Maldonado, Ahmad Yaman Kayali, Wataru Yamazaki, Varaporn Vuddhakul, Fumio Gondaira, Yoshitsugu Nakaguchi, Mitsuaki Nishibuchi
  • Organizer: 49th Annual Joint Panel Meeting on Cholera & Other Bacterial Enteric Infections Panel Meeting, United States-Japan Cooperative Medical Science Program
  • Place of Presentation: Gainsbill, Florida, USA
  • Year and Date: 2015-01-14 – 2015-01-16
  • Invited
• [Presentation] Detection of Shigatoxin-producing Escherichia coli belonging to clinically important O serotypes by an IMS-LAMP-based method in the retailed beef (2015)
  • Author(s): Ahmad Yaman Kayali, Oscar R. Escalante-Maldonado, Kensuke Ozawa, Fumio Gondaira, Varaporn Vuddhakul, Yoshitsugu Nakaguchi, Mitsuaki Nishibuchi
  • Organizer: 49th Annual Joint Panel Meeting on Cholera & Other Bacterial Enteric Infections Panel Meeting, United States-Japan Cooperative Medical Science Program
  • Place of Presentation: Gainsbill, Florida, USA
  • Year and Date: 2015-01-14 – 2015-01-16
  • Invited
• [Presentation] Development of trh gene LAMP primers and establishment of IMS-LAMP based method for detection of pathogenic Vibrio parahaemolyticus in seafood (2014)
  • Author(s): Oscar R. Escalante-Maldonado, Ahmad Yaman Kayali, Sawa Yamada, Wataru Yamazaki, Fumio Gondaira, Varaporn Vuddhakul, Yoshitsugu Nakaguchi, and Mitsuaki Nishibuchi
  • Organizer: 第67回日本細菌学会関西支部総会（兵庫医科大学西宮キャンパス、兵庫県西宮市）
  • Place of Presentation: 兵庫医科大学西宮キャンパス、兵庫県西宮市
  • Year and Date: 2014-11-22 – 2014-11-22
• [Presentation] Prevalence of Shigatoxin-producing Escherichia coli belonging to enterohemorrhagic O serotypes (O157 and non-O157) in the beef marketed in southern Thailand in years 2013 and 2014 (2014)
  • Author(s): Ahmad Yaman Kayali, Oscar R. Escalante-Maldonado, Kensuke Ozawa, Fumio Gondaira, Varaporn Vuddhakul, Yoshitsugu Nakaguchi, and Mitsuaki Nishibuchi
  • Organizer: 第67回日本細菌学会関西支部総会（兵庫医科大学西宮キャンパス、兵庫県西宮市）
  • Place of Presentation: 兵庫医科大学西宮キャンパス、兵庫県西宮市
  • Year and Date: 2014-11-22 – 2014-11-22
• [Presentation] Prevalence of pathogenic strains of Vibrio parahaemolyticus in popular shellfish (molluscan bivalves) marketed in southern Thailand in the 2014 study as demonstrated by a sensitive detection method. 「2014年にタイ南部で人気のある市販二枚貝を対象に高感度検出法を用いて実施した調査において高頻度に検出した病原性腸炎ビブリオ」 (2014)
  • Author(s): Oscar R. Escalante-Maldonado、Ahmad Yaman Kayali、山田佐和、山崎渉、権平文夫、Varaporn Vuddhakul、中口義次、西渕光昭
  • Organizer: 第48回腸炎ビブリオシンポジウム
  • Place of Presentation: 北海道 函館市
  • Year and Date: 2014-11-12 – 2014-11-13
• [Presentation] Analysis of the mechanism of glucose metabolism inhibition, as a cause of hypoglycemia, in falciparum malara using a systems biology approach (2014)
  • Author(s): Koichi Shirakawa, Mitsuaki Nishibuchi
  • Organizer: The 55th Annual Meeting of the Japanese Society of Tropical Medicine
  • Place of Presentation: National Center for Global Health and Medicine, Tokyo, Japan
  • Year and Date: 2014-11-01 – 2014-11-03
• [Presentation] [Keynote] Recent Understanding and Progress in the Technologies (2014)
  • Author(s): Mitsuaki Nishibuchi
  • Organizer: 7th International Seminar, Indonesian Society for Microbology 2014
  • Place of Presentation: Padang, Indonesia
  • Year and Date: 2014-10-15
  • Invited
• [Presentation] Confirmation of the Excellence of the LAMP-IMS-combined Detection Method for Enterohemorrhagic Escherichia coli and of the Modified System Using a Dried Reagent Mix (2014)
  • Author(s): Ahmad Yaman Kayali, Oscar R. Escalante-Maldonado, Kensuke Ozawa, Fumio Gondaira, Akira Ohshima, Varaporn Vuddhakul, Yoshitsugu Nakaguchi, and Mitsuaki Nishibuchi
  • Organizer: 日本防菌防黴学会第41回年次大会
  • Place of Presentation: きゅりあん 東京都品川区
  • Year and Date: 2014-09-24 – 2014-09-25
• [Presentation] Improvement of the immunomagnetic separation (IMS)- and Loop-mediated Isothermal Amplification (LAMP)-based Detection Method for Virulent Strains of Vibrio parahaemolyticus from Seafood (2014)
  • Author(s): Oscar R. Escalante-Maldonado, Ahmad Yaman Kayali, Sawa Yamada, Wataru Yamazaki, Fumio Gondaira, Akira Ohshima, Varaporn Vuddhakul, Yoshitsugu Nakaguchi, and Mitsuaki Nishibuchi
  • Organizer: 日本防菌防黴学会第41回年次大会
  • Place of Presentation: きゅりあん 東京都品川区
  • Year and Date: 2014-09-24 – 2014-09-25
• [Presentation] 腸炎ビブリオの病原機構の解析‐I. 腸管上皮細胞層透過活性と溶血毒遺伝子型との関連性 (2014)
  • Author(s): 藤井愛音，國方千菜美，安田仁，中口義次，西渕光昭，末澤千草, 奥田潤
  • Organizer: 第９回日本臨床検査学教育学会学術大会
  • Place of Presentation: 東京都大田区産業プラザ
  • Year and Date: 2014-08-20 – 2014-08-22
• [Presentation] 腸炎ビブリオの病原機構の解析‐Ⅱ. 腸管上皮細胞層透過活性とTTSS遺伝子群との関連性 (2014)
  • Author(s): 末澤千草，坂元真美，藤井愛音，國方千菜美，安田仁，中口義次，西渕光昭，奥田潤
  • Organizer: 第９回日本臨床検査学教育学会学術大会
  • Place of Presentation: 東京都大田区産業プラザ
  • Year and Date: 2014-08-20 – 2014-08-22
• [Presentation] Comparison of two major diarrheagenic Vibrio species, V. cholerae and V. parahaemolytics, for the ecology, epidemiology, and disease prevention in Asia (2014)
  • Author(s): Mitsuaki Nishibuchi
  • Organizer: Bandung Symposium on ASEAN University Network (AUN)- Kyoto University (KU) Student Mobility Program toward Human Security Development
  • Place of Presentation: Institute of Technology Bandung, Indonesia
  • Year and Date: 2014-06-24 – 2014-06-25
  • Invited
• [Presentation] [Keynote] Recent developments in seafood safety with respect to Vibrio parahaemolyticus (2014)
  • Author(s): Mitsuaki Nishibuchi
  • Organizer: VIBRIO2014
  • Place of Presentation: Edingbrgh, Scotland
  • Year and Date: 2014-04-01 – 2014-04-04
  • Invited
• [Remarks] 世界的に重要な腸管病原性細菌を食品から容易に定量検出できる高感度法の開発
  • URL: http://www.cseas.kyoto-u.ac.jp/research/grant-in-aid-funding/grant-in-aid-funding-a/kaken_mitsuaki_nishibuchi/